Gram-negative bacteria can cause diseases of significant public health and economic concern in humans and other animals. Vaccine strategies are being pursued to combat these infections. These strategies are based on the identification of conserved, immunogenic cell surface components; however, the detection of conserved molecules that would confer protection against the vast majority of strains from a single species has proven problematic.
The exterior surface of the outer membrane of all Gram-negative bacteria contains an amphiphillic carbohydrate molecule termed lipopolysaccharide (LPS) that by virtue of its surface location can be considered as a candidate vaccine antigen. As its name suggests this molecule contains a lipid region that anchors the molecule in the outer membrane, by virtue of both ester (O—) linked and amide (N—) linked fatty acids. The lipid A region and specifically the fatty acids are responsible for the endotoxic activity of the Gram-negative bacterium and consists in most species of a disaccharide of glucosamine sugars that are phosphorylated and the ester and amide linked fatty acids as shown in FIGS. 1 and 2.
The core oligosaccharide can be arbitrarily divided into an outer and inner core and is connected to the lipid A region via one or more ketose sugar(s), 2-keto-3-deoxy-octulosonic acid (Kdo). An O-antigenic polymeric repeating unit (O-antigen) can be present or absent beyond the core oligosaccharide of the LPS molecule. The O-antigen is a variable moiety between strains of the same species and is often the antigen responsible for the serotyping schemes adopted to classify a species. Due to its variable nature within most species the O-antigen is not a good vaccine candidate as antibodies directed to one O-antigen will be serotype specific, and not offer protection to other serotypes of the same strain. Similarly the outer core region can be somewhat variable within a species and is also therefore not a good vaccine candidate. However what is arbitrarily termed the inner core oligosaccharide has been found to be conserved within several species, and is the vaccine antigen of choice in this application. Conserved regions of LPS molecules have been identified in the core oligosaccharide of several species, and examples of core oligosaccharide structures are detailed in FIG. 3 below for LPS from the species Neisseria meningitidis, Haemophilus influenzae and Mannheimia haemolytica. For the purposes of this discussion, but not restricted to just these structures, the inner core region for Neisseria meningitidis, Haemophilus influenzae and Mannheimia haemolytica linked to the lipid A region are illustrated in FIGS. 3b-d. However the technology described here would be equally applicable to the other LPS carbohydrate antigens, outer core oligosaccharide and O-antigen.
The endotoxicity of the lipid A region is due to the fatty acid residues. Removal of the ester-linked fatty acids leaves an O-deacylated LPS species that is no longer endotoxic. Removal of all fatty acids i.e. both the amide and ester-linked fatty acids can be performed chemically, but involves harsh conditions which can effect other regions of the LPS molecule. Even the mild chemical conditions employed to effect O-deacylation can effect ester-linked residues elsewhere in the LPS.
LPS based vaccines generally require the removal of fatty acids from the lipid A region of the molecule to reduce the endotoxicity. Preferably, this detoxification step does not modify the carbohydrate epitopes on the LPS molecule, however commonly available techniques do not permit this.
Current methods employed are to chemically O-deacylate LPS producing an O-deacylated LPS molecule (LPS-OH) which can be used either directly or following further modification to conjugate to a suitable protein carrier to produce a glycoconjugate vaccine candidate. Removal of the remaining N-linked fatty acids from LPS-OH would greatly improve conjugation strategies, as this would create a completely water-soluble molecule amenable to all subsequent manipulations. However, chemical methods currently employed to de-N-acylate LPS molecules also modify some residues in the inner core, thus altering the structure of potentially immunogenic epitopes on the LPS molecule. For example the phosphoethanolamine (PEtn) residue of the inner core oligosaccharide of Neisseria meningitidis LPS (FIG. 3ab), a known immunogenic moiety, is sensitive to the chemical conditions required to remove the N-linked fatty acids, thus modifying a conserved residue in the inner core LPS and creating a molecule which is no longer representative of the native LPS structure.